We will extend our present studies of the adrenal cortical hydroxylation system to study the interactions of adrenodoxin reductase, and cytochromes P-450 in steroid hydroxylation. We will use enzymes from beef adrenal mitochondria and microsomes and from cell cultures of an ACTH-sensitive mouse adrenal cortical tumor line. We will use purified enzymes, membranes, and artificial vesicles. In addition, we will conduct studies on initial and intermediate studies of ES complexes in NADPH-requiring flavoproteins and in the bacterial flavoprotein salicylate hydroxylase.